The long-term aim of this project is to delineate the molecular mechanisms which regulate the activity of the mammalian sirtuin, SIRT6, and to explore the therapeutic potential of our observations which suggest that SIRT6 functions as a tumor suppressor gene, and also may act to attenuate age-related pathologies. SIRT6 is one of seven mammalian homologues of the yeast gene, SIR2, which was identified for its ability to regulate lifespan and promote genomic stability. In the absence of SIRT6, SIRT6[-/-] mice develop a premature aging syndrome characterized by genomic instability and shortened lifespan. It is our hypothesis that mammalian life and health-spans are linked to SIRT6 activity, and we propose that understanding the mechanisms which regulate SIRT6 at the cellular level will reveal important insights into mammalian aging and disease. To this end, we will pursue the following specific aims: Aim 1: Identify post-translational mechanisms which regulate SIRT6 activity. Here we will identify factors which regulate Sirt6 at the post-translational level. We hae unveiled new evidence which suggests that Sirt6 is phosphorylated in vivo, and that this modification enhances Sirt6 stability, allowing the protein to promote DNA repair and cell survival in response to oxidative stress. We intend to delineate the pathway which results in Sirt6 phosphorylation and its relevance to aging and age-related disease. Aim2: Delineate the transcriptional mechanisms that regulate SIRT6 expression. We and other groups have observed that Sirt6 levels are diminished in aging cells. We have observed a similar phenomenon in senescent and non-cycling cells. It appears that a transcriptional mechanism underlies this depletion of Sirt6. We have cloned the promoter of Sirt6 and intend to identify factors which enhance and repress SIRT6 transcription, and how these factors are altered during aging and certain age-related disease processes. Aim 3: Examine an emerging role for SIRT6 as a tumor suppressor. Cancer is one of the most abundant age-related diseases. Excitingly, we found that SIRT6 overexpression is selectively cytotoxic to the cancer cell lines and not to the non-transformed cell lines we have examined thus far. We intend to identify the mechanism through which SIRT6 mediates the killing of these cancer cells, examine whether SIRT6 is selectively cytotoxic to additional cancer cell lines, and test, using a mouse model, whether SIRT6 overexpression protects against tumorigenesis in vivo.